Deoxyribonuclease Test (DNase Test): Principle, Steps, and Importance
Deoxyribonuclease Test
Deoxy ribonucleic acid is one of the large molecule sized polymer, which is being composed of multiple nucleotides in the form of monomer and it is large in size and it is not able to enter cell membrane of the bacteria directly.
Thus, microorganisms like bacteria produces an enzyme known as Deoxyribonuclease, which helps in breaking down the DNA into smaller monomers, so that the cell membrane can easily absorb or intake it.
The nucleotides thus taken are used for making nucleic acid in the bacteria, and it also acts as a source of nitrogen, carbon and phosphorus.
Some microorganism even produces extracellular DNase which breaks down the larger mass of DNA into smaller monomers, so that it is easy for the taking up the vital nutrients that are necessary for the organism through cell membrane by means of protein transports that are present on the membrane of the cell.
But the degradation of DNA is a virulent factor, which causes serious infections in the hosts cell and it results in various drastically symptoms.
The ability to produce DNase can be used in differentiating a different pathogenic organism. Thus, Deoxyribonuclease is an enzyme which helps in catalyzing the hydrolytic cleavage of phosphodiester bonds, which forms the back bone of the DNA, which results in degradation of the DNA.
Deoxyribonuclease is also considered as one of the types of nuclease, a generic form for enzymes that are capable of hydrolyzing phosphodiester bonds.
DNA ribonuclease test is often called as DNA hydrolysis test or DNase test, which helps in determining the ability of the organism to hydrolyze DNA and to utilize it as a source of energy and carbon for the growth.
What is Deoxyribonuclease Test (DNase Test)?
Deoxyribonuclease test is one of the biochemical tests which is being performed to differentiate the organisms on the basis of their ability to produce the enzyme Deoxyribonuclease.
This test is used presumptively to differentiate one of the species of bacteria known as Staphylococcus aureus, which also plays an important role in producing deoxyribonuclease, the enzyme from the other species of staphylococci.
As Staphylococcus aureus produces heat stable enzyme, a thermo nuclease.
To detect this enzyme, initially the organisms are destroyed using heat and then the free DNase reacts in the medium.
This test also gives positive results for Vibrio, Helicobacter, Moraxella, Aeromonas, Serratia.
Deoxyribonuclease Test Principle
This test is used to determine the ability of a particular organism to produce the enzyme DNase.
DNase are the extracellular endonucleases, which cleaves the DNA and there by releases free nucleotides and the phosphates.
To detect these enzymes, DNase agar is used to detect the hydrolysis of the DNA, without the use of any indicators.
When without using any of the indicator, DNA agar helps us to observe the hydrolysis of DNA by clearing the agar after adding HCL.
This acid helps in precipitating the unhydrolyzed DNA and makes the medium opaque.
Hence, DNase producing the colonies produces, colonies which hydrolyses the DNA and results in clear zone around the growth of bacteria.
When DNase is left to react with methyl green it produces, mint green color by combining with DNA.
On hydrolyzing DNA, complex is released and the methyl green changes into a colorless compound at changes its structure when a pH of about 7.5.
Hence it results in the formation of clear halo like appearance around the areas where the organisms producing DNase are grown.
If toluidine blue O is added to the DNase agar.
It results in the formation of complex in the DNA and changes its structure on hydrolyzing the DNA resulting in bright pink color.
Deoxyribonuclease Test Materials
Media: DNase agar or DNase agar with methyl indicator is generally used.
Reagents: Hydrolytic acid is used only when DNase agar is used without using an indicator.
Supplies used: Inoculating loop, Bunsen burner.
Deoxyribonuclease Test Procedure
Initially the Surface of the agar plates are dried before using, Each plate is divided into sections by drawing the line below the glass plate.
Then agar medium is inoculated by either of the following methods.
Spot Inoculation Method
o Touch the colony of the organism under test using a loop and inoculate it on the small area of the test agar plate, in the center of the marked area in any one of the sections, to form a thick plaque of growth about 5-10mm in diameter after incubating.
o The incubation is done at a temperature of about 37ºC for 18 to 24 hours.
o Band or Streak line inoculation.
o Here heavy inoculum is used and a line of about 3 to 4 com long is drawn from rim to the center of the DNase agar plate.
o Plate is then incubated at a temperature of 37ºC for about 18 to 24 hours.
DNase Agar Test Without Indicator
o The plate is being flood with 1N of hydrochloric acid.
o After flooding, the plate is left to stand for few minutes until the reagent is being absorbed into the plate.
o Excess hydrochloric acid is decanted and the plate is further examined in a dark background.
Deoxyribonuclease Test Result
1. Positive Deoxyribonuclease Test: When DNA is hydrolyzed, methyl green is released which turns the medium colorless around the test organism.
2. Negative Deoxyribonuclease Test: In case of negative result, there will be no degradation of DNA, and the medium remain green without any change or absence of color.
Organisms Tested for Deoxyribonuclease Test
Positive organisms in DNase test:
o Serratia Marcescens
o Staphylococcus aureus
o M. Catarrhalis
o Campylobacter jejuni
DNase test negative organisms:
o Staphylococcus epidermidis
o Neisseria gonorrhoeae
Deoxyribonuclease Test Citations
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